Defining the timeline of periostin upregulation in cardiac fibrosis following acute myocardial infarction in mice

After myocardial infarction (MI), the heart's reparative response to the ischemic insult and the related loss of cardiomyocytes involves cardiac fibrosis, in which the damaged tissue is replaced with a fibrous scar. Although the scar is essential to prevent ventricular wall rupture in the infarction zone, it expands over time to remote, non-infarct areas, significantly increasing the extent of fibrosis and markedly altering cardiac structure. Cardiac function in this scenario deteriorates, thereby increasing the probability of heart failure and the risk of death. Recent works have suggested that the matricellular protein periostin, known to be involved in fibrosis, is a candidate therapeutic target for the regulation of MI-induced fibrosis and remodeling. Different strategies for the genetic manipulation of periostin have been proposed previously, yet those works did not properly address the time dependency between periostin activity and cardiac fibrosis. Our study aimed to fill that gap in knowledge and fully elucidate the explicit timing of cellular periostin upregulation in the infarcted heart to enable the safer and more effective post-MI targeting of periostin-producing cells. Surgical MI was performed in C57BL/6J and BALB/c mice by ligation of the left anterior descending coronary artery. Flow cytometry analyses of cells derived from the infarcted hearts and quantitative real-time PCR of the total cellular RNA revealed that periostin expression increased during days 2–7 and peaked on day 7 post-infarct, regardless of mouse strain. The established timeline for cellular periostin expression in the post-MI heart is a significant milestone toward the development of optimal periostin-targeted gene therapy.


In-vitro study of GF-activated cardiac fibroblasts
Production of cardiac fibroblasts from healthy mice and cell culturing 10-14-week-old C57BL/6J healthy male mice (purchased from Envigo (Jerusalem, Israel)) were euthanized with isoflurane and dissected for their heart extraction. The hearts were washed in cold PBS while applying successive pressings to remove the excessive blood. Aorta and atriums were removed, and each heart was minced into pieces measuring ~2 mm and suspended in a 3-ml enzyme solution of 1 mg/ml Liberase® Thermolysin High (Sigma-Aldrich-Merck, Rehovot, Israel) and 10 μM CaCl2 in HBSS. The entire contents were pipetted 12 times using a 5-ml pipette, and put in an orbital incubator shaker for 15 min (85 RPM, 37 °C). Pipetting and shaking were repeated twice more for a total shaking time of 45 min. Next, the solution was pipetted 30 more times using a 1000-μl pipette passed through a 40-μm filter and added 2 to 5 ml of complete fibroblasts-growth culturing medium (88% high glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) Penicillin-Streptomycin-Neomycin (P-S) and 1% (v/v) L-glutamine 200 mM (L-Glu)). The mixture was centrifuged at 1000g for 5 min at 21 °C, the supernatant was aspirated and the cell pellet was resuspended with 10 ml of complete growth medium and seeded in a 10-ml culture plate coated with 0.1% (w/v) gelatin. 24 h after cell seeding, the medium was replaced to remove dead cells and tissue debris, after which the medium was replaced every 2-3 days. Cells were kept incubated in 5% CO2 and ambient O2. When they reached 70-80% confluency, cells were split using trypsin-EDTA to avoid cell contact inhibition and quiescence. PBS, HBSS and cell culturing reagents were all purchased from Biological Industries (BI, Kibbutz Beit- Haemek, Israel).

Growth Factor (GF) Activation
First-and third-passage primary cardiac fibroblasts (PCFs) were seeded to obtain a cell density of 4.5×10 4 cells/well in 0.1% (w/v) coated gelatin in 12-well-plates, after which they were left for overnight incubation to allow them to attach in a monolayer. After 20 h, the culture medium was replaced with a starvation medium, i.e., a low serum medium of 97% (v/v) DMEM supplemented with 1% (v/v) FBS, 1% (v/v) P-S and 1% (v/v) L-Glu (1 ml per well). 24 h later, the starvation medium was removed, the cells were washed once with PBS, and for the final step, the cells were then exposed to an activation medium, i.e., a low serum medium supplemented with growth factors: 10 ng/ml Mouse-TGF-β1 (BioLegend, Petach-Tiqva, Israel); 20 ng/ml Mouse-PDGF-B (BioLegend). Cells were incubated for either 24 or 48 h.

Gene Expression Analysis
RNA Extraction of cultured PCFs: Culture medium was removed from cells, which were then washed once with cold PBS. Approximately 1×105 PCFs (from growthfactor activation) were pelleted and their mRNA was extracted using cell lysis buffer, homogenization, ethanol precipitation and RNA mini-column for RNA binding and  Table S1. GAPDH was used as the reference housekeeping gene (endogenous control for normalization) to calculate the ΔCt and the fold change of mRNA expression (ΔΔCt) relative to the negative control group. Each 10 µl of reaction medium contained 2 µl cDNA (5 ng). Activated fibroblasts [9] ; myofibroblasts [9] Mouse Mm01546133_m1 Acta2 Actin; alpha 2; smooth muscle; aorta

Gene Expression Analysis
Reverse transcription and quantitative real-time PCR (qPCR) were performed as described in section 1.1 above. HPRT was used as the reference housekeeping gene.

In-Vitro studies of Activated Fibroblasts
An in-vitro cell culture system was chosen as the first step in mimicking this described post-MI activated profile of cardiac fibroblasts and to confirm the increase in the production of periostin (and other fibrotic markers). Gene expression analysis was conducted on cultured primary cardiac fibroblasts (PCFs) isolated from healthy mice, which were then activated with growth factors present in organ fibrosis: TGF-β1, considered the most potent stimulator of myofibroblast differentiation [13] [14] , and PDGF-BB, known for its mitogenic effect on cells [15] . test, means ± SD, **** = p < 0.0001, *** = p < 0.001, ** = p < 0.01, * = p < 0.05).

Post-MI periostin expression
To establish a representative, post-MI timeline of periostin upregulation in cardiac fibroblasts, production and identification of cardiac fibroblasts was executed on MIinduced wild-type C57BL/6J female and male mice at different time points after the MI (Fig. S3). Healthy mice that did not undergo the MI surgery used as references to the standard protein expression in non-pathological cardiac tissue. Flow cytometry results depict an increase in periostin expression levels in the infarcted heart (the part of the produced living cell population that comprises periostin-expressing cells) starting on day 2 after MI and lasting up to 7 days after MI (Fig. S3A1-E1) that is indicative of fibroblast activation. In line with the timeline found via gene analysis (Fig. 1A), 7 periostin expression began to decline after day 7 (Fig. S3F1). Furthermore, periostin's expression levels peaked 5-7 days after the MI with a considerable peak on day 7 ( Fig.   S3C1-E1), hereby reinforcing the specified timeframe of periostin major upregulation in the infract heart (Fig. S3G). Expectedly, since periostin is known to be expressed after MI by activated fibroblasts [ [16] , measurements of periostin expression in cells that also express MEFSK4 show that the timeline of periostin expression is preserved: five days after the MI, the population of cells expressing both MEFSK4 and periostin was markedly enhanced (Fig. S3C3 compared to Fig. S3A3), continuing its growth to seven days after the MI (Fig. S3E3). On day 14, the population size was again diminished (Fig. S3F3), overall correlating with the above-mentioned timeline of periostin upregulation in the infracted heart ( they are assumed to originate from endothelial cells that have undergone epithelial to mesenchymal transition (EMT) [3][16] [17] , making them somewhat different from resident fibroblasts of the myocardium which are derived from the mesenchymal linage and recognized by MEFSK4 [3][17][18] [19] . Since periostin is found on these cells from early stages of healthy cardiac development [20] , this may also explain periostin expression in the absence of the MI trigger (Fig. S3A1, A3). Similar to periostin, MEFSK4 expression escalated during the first six days after the MI (Fig. S3A2-D2) and peaked on day 7 ( Fig. S3E2). As MEFSK4 was shown to be independent of cell activation [19] , and indeed its upregulation was not disabled by periostin upregulation (Fig. S3E1, E2), this escalation is most likely the outcome of fibroblasts proliferation, known to take place in response to the insult along with periostin expression [21] . Fourteen days after the MI, MEFSK4 expression declined (Fig. S3F2), marking the end of the post-MI upregulation of MEFSK4 (Fig. S3H). The larger MEFSK4+ cell populations compared to those of periostin+ at each specified time point can be explained by the recognition of murine cardiac fibroblasts by the MEFSK4 antibody, regardless of fibroblast activation status.
Insofar as cells were originally extracted from whole ventricles (Fig. 2), non-activated fibroblasts (negative to periostin) were also included in the analyses. Protein expression timeframes inferred from analyses post-MI of the BALB/c mouse strain (Fig. 5F-H) show a strong resemblance to that described in C57BL/6J mice (Fig. S3G-I) G  H  I